The primary aim of the proposed research is to continue investigating the relationship between formation of PAH DNA adducts and tumor initiation in mouse epidermis. During the course of the previous funding period, we identified a PAH, DB[aj]A, that produces tumors with exclusively A182 T transversion mutations in c-Ha-ras similar to that observed with DMBA. In addition, 100% of the tumors analyzed that were initiated with (+)anti-DB[aj]A-diol-epoxide (DE) also had A 182 T transversion mutations in C-Ha-ras. Furthermore, we found that the mutation spectrum of the (+)anti-DB[aj]A-DE in the supF gene is dominated by mutations at dGuo residues. A important question we will address in this renewal application is what is the basis for this selectivity for dAdo mutations in c-Ha-ras when, in fact, both DB[aj]A and (+)anti-DB[aj]A-DE form significant amounts of dGuo as well as dAdo adducts in epidermal DNA. The selectively of mutations in c-Ha-ras could be due to: 1) different inherent mutagenic properties of the N2-dGuo vs No-dAdo adduct formed; ii) different mutagenic potential due to the sequence context of the 12th/13th vs 61st codons; iii) stronger differences in the amount of specific adducts formed at these sites; and v) differential repair. During the previous funding period, we also prepared site-specific adducts, in a bacterial replication vector to allow us to compare the mutagenicity of the (+)anti-DN[aj]A-DE-tran-N2-dGuo and (+)anti-DB[aj]-A- DE-trans-N0-DNA adducts. In this renewal application, we will continue to test the hypothesis that the presence of specific factors other than the inherent mutagenicity of specific adducts contribute to the selectivity of mutations observed in c-Ha-ras with certain PAH. The Specific Aims are: 1) To examine the mutations in the c-Ha-ras gene from a larger number of skin tumors initiated by DB[aj]A and 7- methylbenz[a]anthracene(7-MBA); 2) To determine the mutations in c-Ha-ras early after topical application of DB[aj]A and 7-MBA and prior to promoter treatment (i.e., initiated epidermis) and whether the spectrum of mutations in the same as that found in initiated/hyperplastic skin and skin tumors; 3) To further examine the mutagenic specificity of (+)-anti- DB[aj]A-DE in the supF gene in bacteria; 4) Examine the sequences effects and sequence specificity of binding of (+)anti-DB[aj]A-DE in; i_ oligonucleotides containing specific dGuo or dAdo residues and different 5'or 3' bases; ii) in exons I and II of murine c-Has; and 5) Through the use of site-specific adducts, study: i) the mutagenic potential of specific dGuo and aADo adducts derived from (+)anti-DB[aj]A-DE; ii) biochemical and structural effects of specific adducts on DNA; and iii) the effect of different sequence contexts on the mutagenicity of these adducts.